Nitric oxide (NO) is a versatile free radical that mediates numerous biological functions within every major organ system. An emerging molecular pathway by which NO accomplish functional diversity is the specific modification of protein cysteine residues to form S-nitrosocysteine. This post-translational modification, S-nitrosylation, impacts protein function and location. Despite considerable advances with individual proteins, the biological chemistry, the dependency on specific nitric oxide synthases (NOS) and the structural elements that govern the modification of specific cysteine residues in vivo are vastly unknown. Moreover comprehensive studies exploring protein signaling pathways or interrelated protein clusters that are regulated by S-nitrosylation have not been performed. To provide insights for these important biological questions, sensitive, validated and quantitative proteomic approaches are needed but are not currently available. To this end, during the last funding period we developed and implemented a novel mass spectrometry-based proteomic approach. The new method achieved specific, efficient, complementary and selective identification of the modified cysteine residue. Currently implementation of the method has precisely pinpointed the site of S-nitrosylation in 741 peptides, which were independently matched to 521 proteins in mouse liver, heart, lung brain and thymus. These proteins constitute the largest datasets of endogenous S-nitrosylated proteins to date. Using this robust new methodology we propose to: (1) Define the structural elements that govern the specificity of S-nitrosylation, (2) Elucidate the functional networks and signaling pathways that are influenced by S-nitrosylation and (3) Determine if S-nitrosylation represents the molecular link between eNOS and leptin in the regulation of liver lipid metabolism. By uncovering the endogenous S-nitrosocysteine proteomes of mouse liver, brain, lung and heart and applying multiple analytical and computational tools, the structural properties that govern the specificity and selectivity of S-nitrosylation in vivo will be defined. By identifying the S-nitrosocysteine proteome of mice which do not express S-nitrosoglutathione reductase (GSNOR), the enzyme that metabolizes S-nitrosoglutathione (GSNO), we will elucidate the structural elements governing the in vivo GSNO-mediated S-nitrosylation. Functional pathway and network analyses in conjunction with quantitative assessment of S-nitrosoproteomes derived from endothelial NOS (eNOS), neuronal NOS (nNOS) and GSNOR null mice will test NOS specific functional regulation in signaling cascades within and across the four different organs. A novel hypothesis linking eNOS-mediated S-nitrosylation with leptin in the regulation of liver lipid metabolism will be explored. Overall the comprehensive large-scale study of protein structures and functional pathways will significantly improve our appreciation of S-nitrosylation in nitric oxide biology.